Dendritic cells (DCs) are recognized to be powerful antigen presenting cells for inducing cellular immunologic responses in humans, and play a key role in eliciting effective anti-tumor immune responses. DCs prime both CD8+cytotoxic T-cell (CTL) and CD4+T-helper (Th 1) responses. DCs are capable of capturing and processing antigens, and migrating to the regional lymph nodes to present the captured antigens and induce T-cell responses. In humans, DCs are a relatively rare component of peripheral blood (<1%), but large quantities of DCs can be differentiated from CD34+precursors or blood monocytes utilizing expensive cytokine cocktails. Alternatively, by treating an extracorporeal quantity of blood using a process referred to herein as trans immunization, a large number of immature DCs can be induced to form from blood monocytes without the need for cytokine stimulation. These immature DCs can internalize and process materials from disease effectors, such as antigens, DNA or other cellular materials, to induce cellular immunologic responses to disease effectors. By exposing increased numbers of dendritic cells to cellular material, such as for example antigens from tumor or other disease-causing cells, followed by reintroduction of the loaded dendritic cells to the patient, presentation of the cellular material to responding T-cells can be enhanced significantly.
For example, one in vitro method previously used involves culturing blood mononuclear leukocytes for six to eight days in the presence of granulocyte-monocyte colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) to produce large numbers of dendritic cells. These cells can then be externally loaded with tumor-derived peptide antigens for presentation to T-cells. Alternatively, the dendritic cells can be transduced to produce and present these antigens themselves. Expanding populations of dendritic cells transduced to produce and secrete cytokines which recruit and activate other mononuclear leukocytes, including T-cells, has shown some clinical efficacy in generating anti-tumor immune responses.
However, transducing cultivated dendritic cells to produce a particular generic antigen and/or additional cytokines is labor intensive and expensive. More importantly, when used to treat a disease such as cancer, this procedure likely fails to produce and present those multiple tumor antigens that may be most relevant to the individual's own cancer. Several approaches have been proposed to overcome this problem. Hybridization of cultivated autologous dendritic cells with tumor cells would produce tetraploid cells capable of processing and presenting multiple unknown tumor antigens. In a second proposed approach, acid elution of Class I and Class II major histocompatability complexes (MHC) from the surface of malignant cells would liberate a broad spectrum of tumor-derived peptides. These liberated peptides could then be externally loaded onto MHC complexes of autologous cultivated dendritic cells.
Because there are limitations to each of these approaches, an improved method of producing functional antigen presenting dendritic cells and for loading the dendritic cells with cellular material from disease causing agents is desirable. In U.S. Pat. Nos. 6,524,855, 6,607,722 and 7,109,031, the entire contents of each of which are hereby incorporated by reference, methods of producing increased numbers of functional dendritic cells are described. The methods described in these patents generally involve exposure of blood monocytes to internal surfaces of a plastic treatment device. As the blood monocytes flow past the plastic surface of the treatment device, interaction with the plastic surface induces differentiation of the monocytes into dendritic cells. The dendritic cells may then be incubated with apoptotic disease cells to produce antigen presenting dendritic cells. These methods typically produce mature dendritic cells which can be used to enhance the immune response to disease cells.
In U.S. patent application Ser. No. 10/290,802, the entire contents of which are hereby incorporated by reference, methods of producing immunosuppressive dendritic cells are described. Dendritic cells are produced by treating monocytes in a plastic treatment device as described above. The maturation of the dendritic cells is truncated at a stage where the dendritic cells are immunosuppressive.
While the methods described in these references are effective in producing relatively large numbers of dendritic cells, it would be desirable to have a process that further increases the number of dendritic cells produced from a quantity of a patient's blood. It would also be desirable to control the process to produce the desired types of dendritic cells (i.e. immune enhancing or immunosuppressive).
Accordingly, it is an object of the present invention to provide methods to produce large quantities of dendritic cells having desired functionality as immune enhancing or immunosuppressive. Other objectives and advantages of the present invention will be apparent to one skilled in the art based upon the description of preferred embodiments set forth below.